This proposal addresses the great need for definitive evidence to test the prevalent dogma that Foxp3+/Tregulatory cells are essential for the acceptance of physiologically relevant MHC incompatible organ grafts. MHC-mismatched mouse renal allografts are often accepted without specific therapy in certain strain combinations, and can promote acceptance of other tissues from the same donor. Heart allografts do not have this property, suggesting an organ specific feature of the kidney. Despite demonstration of this phenomenon over 30 years ago, the mechanisms by which kidneys promote tolerance to other tissues have not been established. This project will test the role of Foxp3+ T regulatory cells and indoleamine 2,3-dioxygenase (IDO) in the pathogenesis of acceptance of mouse renal allografts, using full MHC mismatched combinations that are known to accept 33-80% of the grafts. Particular attention will be paid to interactions of Foxp3 cells with renal tubules, since Foxp3 cells concentrate in this site. Foxp3+ cells will be deleted systemically and in the graft with using the diphtheria toxin receptor (DTR) transgenic mouse with DTR expressed from the foxp3 promoter in conjunction with GFP. Retransplant experiments will test whether intragraft T cells are sufficient to reject the graft once intragraft Foxp3 cells are removed. Interaction of Foxp3+ cells with other T cells, dendritic cells and tubular cells in the renal grafts will be assessed with a real time confocal, multiphoton endomicroscope developed in our laboratory using transgenic mice expressing GFP or other fluorescent marker proteins in these cells. This system permits repeated observations of the same graft over time, live video recording, delineation of three different cell types and computer analysis of cell motility and interactions. Biopsies of renal allografts will be analyzed for cellular content (Foxp3+, Tbet+, IDO expression) in addition to standard Banff scoring as done in the human to determine which features correlate with later graft acceptance, using whole slide imaging technology and morphometric immunohistochemistry. The role of intragraft TGF&#946; activation, IL-6 and IDO production and CD103 expression in the generation of Treg will be assessed. Functional studies of infiltrating cells will be compared with those in the spleen for ELISPOT direct and indirect reactivity to the donor and inhibition by Foxp3+ cells.